The activity of bovine liver glutamate dehydrogenase is affected by a variety of compounds including purine nucleotide, as exemplified by GTP which inhibits, ADP and cAMP which activate, and DPNH which inhibits by binding to a site distinct from the active site. The enzyme is unusual in its relative lack of specificity for coenzyme, accepting either DPN or TPN in the catalytic oxidative deamination of glutamate. Although considerable effort has been devoted to the study of the structure of glutamate dehydrogenase and its amino acid sequence has been determined, the coenzyme and purine nucleotide regulatory sites have not been located within the sequence of the enzyme. It is proposed to use several adenine and guanine nucleotide analogues, most of which have recently been synthesized in this laboratory, as affinity labels of the distinct catalytic and regulatory sites of glutamate dehydrogenase. These compounds have alkylating functional groups capable of reacting covalently with amino acid residues of the enzyme. In the case of each specific modification, the kinetic and binding properties of the altered enzyme will be examined to elucidate its functional aberration. With radioactive reagents, labeled enzyme will be prepared; peptides will subsequently be isolated and the particular modified amino acid within the sequence will be identified. Advantage will be taken of the special properties of nucleoside-linked peptides in their purification. By using affinity labeling reagents which incorporate functional groups at various distinct and defined positions of the ribose or purine ring, it should be possible to map the regions of glutamate dehydrogenase which contribute to the various nucleotide sites. It is anticipated that the approaches developed in using these compounds to probe the nucleotide sites of glutamate dehydrogenase will have implications for the exploration of purine nucleotide sites in a wide range of other proteins.